阅读说明：本技术 一种山药多糖、其制备方法及其用途 (Chinese yam polysaccharide, preparation method and application thereof ) 是由 丁侃 陈霞 潘浩宇 张春倩 于 2020-04-10 设计创作，主要内容包括：本发明涉及一种山药多糖、其制备方法及其用途,具体涉及一种从山药当中提取得到的山药多糖、其制备方法,以及其在制备预防和/或治疗肠道菌群紊乱的药物或保健品中的用途。所述山药多糖是一种1,4-α-半乳糖醛酸聚糖,具有如下结构：[→4)-α-D-GalAp(1→4)-α-D-GalAp(1→]<Sub>n</Sub>,且重均分子量范围为1-65kDa。该多糖可以缓解复急性腹泻症状,具有潜在的治疗急性腹泻,调节肠道紊乱的效果,有望开发成为一种治疗肠道紊乱疾病的糖类药物。(The invention relates to Chinese yam polysaccharide, a preparation method and application thereof, in particular to Chinese yam polysaccharide extracted from Chinese yam, a preparation method and application thereof in preparing a medicine or health care product for preventing and/or treating intestinal flora disorder, wherein the Chinese yam polysaccharide is 1,4- α -galacturonic acid glycan and has the following structure [ → 4) - α -D-GalAp (1 → 4) - α -D-GalAp (1 → 4)] n And a weight average molecular weight in the range of 1-65 kDa. The polysaccharide can relieve the symptoms of acute diarrhea, has potential effects of treating acute diarrhea and regulating intestinal tract disorder, and is expected to be developedBecomes a carbohydrate medicine for treating intestinal disorder diseases.)

1. A yam polysaccharide is a 1, 4-alpha-galacturonic acid glycan and has the following structure:

[→4)-α-D-GalAp(1→4)-α-D-GalAp(1→]_n

and the weight average molecular weight is in the range of 1-65 kDa.

2. A method for preparing the yam polysaccharide of claim 1, comprising:

(1) and (3) extracting polysaccharide: extracting rhizoma Dioscoreae with boiling water to obtain extractive solution, concentrating the extractive solution, precipitating with ethanol, centrifuging, and collecting precipitate to obtain crude polysaccharide;

(2) and (3) purifying polysaccharide: subjecting crude polysaccharide to DEAE Sepharose^TMSeparating by a Fast Flow anion exchange column, and collecting elution components of 0.2M NaCl eluent to obtain secondary crude polysaccharide; and further purifying the secondary crude polysaccharide by a Sephacryl S-300HR gel column to obtain the yam polysaccharide.

3. The method according to claim 2, wherein the anion exchange column separation of step (2) is performed by gradient elution using deionized water, 0.1M NaCl solution and 0.2M NaCl solution in sequence, and the elution components of the 0.2M NaCl eluate are collected.

4. The method of claim 2, wherein: and (3) when the Sephacryl S-300HR gel column in the step (2) is further purified, eluting by using 0.2M NaCl eluent, and collecting the obtained elution component to obtain the yam polysaccharide.

5. The method as claimed in claim 2, wherein in the step (1), the yam is soaked in ethanol for 3-10 days before the extraction with boiling water, and then dried in the air.

6. The method according to claim 2, wherein in the step (1), the ethanol used for precipitation is ethanol with a volume fraction of 95%, and the volume of the ethanol used for precipitation is 3-6 times of that of the concentrated solution.

7. The method according to claim 2, wherein in the step (1), the concentrate is centrifuged before ethanol precipitation, and then the centrifuged supernatant is dialyzed.

8. The process according to claim 2, wherein the crude polysaccharide obtained in step (1) is washed before the purification of the polysaccharide in step (2), with alternating washes of ethanol and acetone.

9. A pharmaceutical composition characterized by: comprises the yam polysaccharide of claim 1 and pharmaceutically acceptable auxiliary materials.

10. Use of the yam polysaccharide of claim 1 or the pharmaceutical composition of claim 9 for the preparation of a medicament or health product for the prevention and/or treatment of a disturbance of intestinal flora.

Technical Field

The invention relates to yam polysaccharide, in particular to yam polysaccharide DOP0.2-S-3 extracted from yam (Dioscorea opposita Thunb.), a preparation method thereof and application thereof in preparing a medicament or health-care product for preventing and/or treating intestinal flora disorder.

Background

The steady state and diversity of intestinal flora are closely related to host cell proliferation, nerve signal pathways and the like. In the adult, the intestinal microbiota forms a significant core microbiota, thereby reducing the influence of internal and external environmental changes and maintaining the balance of the body. However, in the aging process of human body, the decrease of exercise, the change of nutrition, the change of medicine and the living condition all affect the microorganism and the metabolite thereof, and bring about the change of physiology and immunity.

Chinese yam (Rhizoma Dioscoreae) is dried rhizome of Dioscorea opposita Thunb (Dioscorea opposita Thunb.) of Dioscoreaceae, and is a Chinese medicinal preparation used as both medicine and food. According to the records of the compendium of materia medica, Chinese yam is sweet, warm, mild and nontoxic in nature and mainly treats the symptoms of weakness of spleen and stomach, damp-heat and deficiency-heat, initial pyogenic infections and the like. The rhizoma Dioscoreae contains chemical components such as saponin, choline, starch, amino acids, etc.

Disclosure of Invention

The invention uses a simple and effective process and method for extracting and purifying plant polysaccharide, uses Chinese yam from Hebei producing areas as raw materials to obtain a mixed polysaccharide component, and further performs purification to obtain uniform polysaccharide DOP 0.2-S-3. In vivo experiments prove that the polysaccharide can relieve acute diarrhea symptoms caused by compound antibiotics (clindamycin, ampicillin, ceftriaxone sodium and vancomycin). Therefore, the polysaccharide has potential effects of treating acute diarrhea and regulating intestinal disorders, and is expected to be developed into a saccharide medicine for treating intestinal disorder diseases.

The invention provides a yam polysaccharide DOP0.2-S-3, which is 1, 4-alpha-galacturonic acid glycan and has the following structure:

[→4)-α-D-GalAp(1→4)-α-D-GalAp(1→]_n

the weight average molecular weight is in the range of 1-65 kDa.

The invention also provides a preparation method of the yam polysaccharide DOP0.2-S-3, which comprises the following steps:

(1) and (3) extracting polysaccharide: extracting rhizoma Dioscoreae with boiling water to obtain extractive solution, concentrating the extractive solution, precipitating with ethanol, and collecting precipitate to obtain rhizoma Dioscoreae crude polysaccharide DOP;

(2) and (3) purifying polysaccharide: crude polysaccharide DOP by DEAE Sepharose^TMSeparating by Fast Flow anion exchange column, collecting the eluted component of 0.2M NaCl eluate to obtain secondary crude polysaccharide DOP0.2 of rhizoma Dioscoreae, and further purifying the secondary crude polysaccharide DOP0.2 by Sephacryl S-300HR gel column to obtain the polysaccharide DOP0.2-S-3 of rhizoma Dioscoreae.

Preferably, in the step (1), before the extraction with boiling water, the yam is soaked in ethanol for 3-10 days (for example, 7 days), and then dried in the air;

preferably, in the step (1), ethanol used for precipitation is ethanol with a volume fraction of 95%; the volume of ethanol used for precipitation is 3-6 times (for example, 5 times) of that of the concentrated solution;

preferably, in the step (1), before ethanol precipitation, the concentrated solution is centrifuged, and then the centrifuged supernatant is dialyzed, wherein the dialysis time is preferably 24-48 h;

preferably, the crude polysaccharide obtained in step (1) is washed before the purification of the polysaccharide in step (2), with alternating washes of ethanol and acetone, each wash 2-4 times;

preferably, in step (1), the precipitate is dried, e.g. lyophilized, after it has been collected or after it has been washed alternately with ethanol and acetone;

preferably, during the anion exchange column separation in the step (2), deionized water, 0.1M NaCl solution and 0.2M NaCl solution are sequentially used for gradient elution, and elution components of the 0.2M NaCl eluent are collected;

preferably, when the Sephacryl S-300HR gel column in the step (2) is further purified, 0.2M NaCl eluent is used for elution, and the collected elution component is yam polysaccharide DOP 0.2-S-3.

The identification of the obtained yam polysaccharide DOP0.2-S-3 comprises the determination of purity and molecular weight, and then the structural characteristics of the yam polysaccharide DOP are analyzed by adopting methods such as the determination of sugar composition, nuclear magnetic resonance and the like.

The invention also provides a pharmaceutical composition which comprises the yam polysaccharide DOP0.2-S-3 and pharmaceutically acceptable auxiliary materials.

The invention also provides application of the yam polysaccharide DOP0.2-S-3 or the pharmaceutical composition in preparing a medicament or a health-care product for preventing and/or treating intestinal flora disorder. Such disorders of the intestinal flora include, for example, acute diarrhea. The acute diarrhea is caused by antibiotic complex (clindamycin, ampicillin, ceftriaxone sodium and vancomycin).

Drawings

FIG. 1 is a purity chart of high performance liquid chromatography of yam polysaccharide DOP0.2-S-3 prepared in preparation example 1.

FIG. 2 is a drawing showing that the yam polysaccharide DOP0.2-S-3 obtained in preparation example 1¹H NMR (A) and¹³c NMR spectrum (B).

FIG. 3 is a line graph showing the effect of the yam polysaccharide DOP0.2-S-3 prepared in preparation example 1 on the body weight of mice in a model of acute diarrhea caused by antibiotics.

FIG. 4 is a scattergram of the number of times of defecation of mice in a mouse acute diarrhea model caused by antibiotics of the yam polysaccharide DOP0.2-S-3 prepared in preparation example 1, wherein A is a count result on day 7 and B is a count result on day 14.

FIG. 5 is a bar graph showing the water content of feces of mice in a mouse acute diarrhea model caused by antibiotics to which yam polysaccharide DOP0.2-S-3 prepared in preparation example 1 is added, wherein A is a detection result on day 7, and B is a detection result on day 14.

Detailed Description

Preparation example 1 extraction, separation, purification and structural characterization of polysaccharide component DOP0.2-S-3 of Yam

(1) Extraction and separation of polysaccharides

Extracting rhizoma Dioscoreae polysaccharide with boiling water for several times, and detecting sugar content of the extractive solution with sulfuric acid-phenol method until sugar reaction is not obvious. Mixing extractive solutions, concentrating to small volume, and centrifuging to remove precipitate. The supernatant was dialyzed against convective water for two days. The internal liquid for dialysis is prepared from 95% ethanol by volume ratio of extract to ethanol of 1: 3(v/v) and standing overnight. Removing the excessive ethanol on the upper layer, centrifuging the precipitate, alternately washing the precipitate with ethanol and acetone for three times, and drying to obtain crude polysaccharide DOP (4.4%) extracted from rhizoma Dioscoreae water.

(2) Purification of polysaccharides

Dissolving 7.3g of crude polysaccharide in 80mL of deionized water, stirring overnight, centrifuging, collecting supernatant, and loading on DEAE Sepharose^TMThe Fast Flow anion exchange column was subjected to gradient elution with deionized water and NaCl solutions (0.1M, 0.2M) of different concentrations, the Flow rate was controlled at 13mL/15min, and the eluate was collected with an automatic collector. mu.L of each tube was developed by the sulfuric acid-phenol method and the absorbance thereof was measured at 490nm with a microplate reader, and the elution curve was plotted using the absorbance and the elution volume. Collecting separated polysaccharide according to elution curve, concentrating under reduced pressure, dialyzing, freeze drying to obtain 0.2M NaCl eluate, and drying to obtain secondary crude polysaccharide DOP0.2(261 mg).

Dissolving 85mg of secondary crude polysaccharide DOP0.2 in 3mL of deionized water, centrifuging (4,000r/min), collecting supernatant, loading on Sephacryl S-300HR gel column, eluting with 0.2M NaCl eluent at flow rate of 5mL/15min, and collecting with automatic collector. Coloring with sulfuric acid phenol method, detecting absorbance with enzyme labeling instrument, drawing elution curve, collecting the required separated components, concentrating, dialyzing, and freeze drying to obtain uniform rhizoma Dioscoreae polysaccharide DOP0.2-S-3(21mg, 24.7%).

(3) Structural identification of polysaccharides

Polysaccharide DOP0.2-S-3 in series Ultrahydrogel^TM2000(7.8mm × 300mm, exclusion limit 5 × 10 mm⁴-10×10⁶Da) and Ultrahydrogel^TM500(7.8mm × 300mm, exclusion limit 1 × 10 mm⁴-4×10⁵Da) analysis the characteristic pattern on the gel column is shown in figure 1, and the chromatographic conditions are as follows: mobile phase: 0.1M NaNO₃(ii) a Flow rate: 0.5 mL/min; column temperature: 25 ℃; agilent 1260 liquid chromatograph; a detector: a differential detector and an ultraviolet detector. The weight average molecular weight of the sample was calculated from the polysaccharide standard curve: respectively dissolving standard dextran series (180Da, 667Da, 6,000Da, 11,300Da, 21,700Da, 48,800Da, 113,000Da, 210,000Da, 393,000Da, 805,000Da) with known molecular weight in mobile phasePreparing a solution with the concentration of 2mg/mL, centrifuging, taking supernatant, and carrying out automatic sample injection analysis. Standard curves were drawn by GPC-specific software.

Taking rhizoma Dioscoreae polysaccharide component sample DOP 0.2-S-335 mg, adding D₂O0.5 mL, added 2.5. mu.L acetone as internal standard: (_H＝2.29ppm，_C31.5ppm) were measured on a Bruker AVANCE III 500M nmr spectrometer at 25 ℃ in each case on one-dimensional nmr spectra, the results being shown in fig. 2. A in FIG. 2 represents a polysaccharide¹H NMR spectrum, chemical shifts of H1-H5 in galacturonic acid glycan were 5.13ppm, 3.82/3.84ppm, 4.05/4.07ppm, 4.48ppm and 4.83ppm, respectively. B in FIG. 2 represents a polysaccharide¹³C NMR spectra and chemical shifts of C1-C6 were 100.24ppm, 69.34ppm, 70.04ppm, 79.14ppm, 72.49ppm and 176.55ppm, respectively. The polysaccharide DOP0.2-S-3 has the specific structure as follows:

[→4)-α-D-GalAp(1→4)-α-D-GalAp(1→]_n

the weight average relative molecular mass of DOP0.2-S-3 in the yam polysaccharide component sample is 14kDa by High Performance Gel Permeation Chromatography (HPGPC).

Test example 1 Activity of polysaccharide component DOP0.2-S-3 of Yam for regulating growth of intestinal flora

(1) Detection of influence of polysaccharide component DOP0.2-S-3 of Chinese yam on animal weight by antibiotic-induced acute diarrhea model

The animal experiment was approved by animal ethics committee of Shanghai pharmaceutical research institute of Chinese academy of sciences in compliance with the animal management and use guidelines, and was approved by IACUC approval No. 2019-02-DK-74.

20 four week-old male C57BL/6 mice were purchased from Shanghai Ling Biotech, Inc. Animals were randomly divided into three groups, 6 control groups, 6 model groups, 8 experimental groups, and one litter of 4-6 animals were kept, and mice had free access to sterile drinking water and food. The animals were kept in a constant temperature (25 ℃. + -. 2 ℃) light cycle (12 hours light, 12 hours dark) environment and the experiment was started after one week of acclimatization.

The water in the model group and the experimental group was added with mixed antibiotics (clindamycin: ampicillin: ceftriaxone sodium: vancomycin: 3: 1: 1) at a concentration of 500ug/mL for one week. After one week, sterile drinking water is replaced for one week. 30mg/kg DOP0.2-S-3 polysaccharide was administered to the animals of the experimental group once a day for fourteen days, 0.2mL each time, from the first day of the experiment, and the model group and the control group were administered with an equal volume of physiological saline. Animal body weights were measured three times per week.

Results as shown in figure 3, the animal weights in the model group (15.98 ± 0.22) were significantly lower than those in the control group (18.78 ± 0.35, × p <0.001) and experimental group (18.41 ± 0.33, × p <0.001) after four days of antibiotic treatment. At the end of the animal experiment, the body weight of the model group (20.08 ± 0.17 × p <0.001) and the body weight of the experimental group (21.13 ± 0.35 × p <0.05) were significantly reduced compared to the control group (22.56 ± 0.34), but the body weight of the experimental group was higher than that of the model group (p ═ 0.0504), indicating that the polysaccharide DOP0.2-S-3 could reduce the symptoms of antibiotic-induced acute diarrhea.

(2) Defecation frequency counting detection of DOP0.2-S-3 influence on defecation frequency

Each animal was placed individually with sterile, transparent rat chow, and the number of stools was counted within five minutes and ten minutes for each animal, and counted on the seventh and fourteenth days. The results are shown in FIG. 4.

As can be seen from a in fig. 4, on day seven, the number of stools in the model group was significantly increased within five minutes (5.5 ± 0.5, × p <0.01) and ten minutes (9.0 ± 0.61, × p <0.001) compared to the control group (2.83 ± 0.17, 4.83 ± 0.31), and the number of stools in the experimental group was significantly decreased within five minutes (2.87 ± 0.44, × p <0.01) and ten minutes (4.62 ± 0.50, × p <0.001) compared to the model group, with no significant difference between the control group and the experimental group. As can be seen from B in fig. 4, on the fourteenth day, the number of stools in the model group was significantly increased within five minutes (4.00 ± 0.26, × p <0.001) and ten minutes (7.33 ± 0.42, × p <0.01) compared to the control group (0.40 ± 0.40,3.4 ± 0.92), the number of stools in the experimental group was significantly decreased within five minutes (2.25 ± 0.41, × p <0.01) and ten minutes (4.00 ± 0.46, × p <0.01) compared to the model group, and there was no difference in the number of stools between the control group and the experimental group within ten minutes. It is shown that the polysaccharide DOP0.2-S-3 can reduce the increase of defecation frequency caused by antibiotic-induced acute diarrhea.

(3) Detection of Water content in animal feces

Each animal was placed individually with sterile, clear rat chow and the entire feces of the animals were collected over ten minutes and examined on the seventh and fourteenth days. Weighing fresh excrement, placing the fresh excrement in an oven at 100 ℃ for drying for 24 hours, and weighing the dry weight of the excrement. The calculation formula is as follows: stool water content (%) - (fresh stool weight-dry stool weight)/fresh stool weight. The results of the experiment are shown in FIG. 5.

As can be seen from a of fig. 5, the fecal water content of the model group (79.28 ± 0.98, # p <0.001) was significantly increased compared to the control group (59.86 ± 1.61) at the seventh day, the fecal water content of the experimental group (73.76 ± 0.52, # p <0.01) was significantly decreased after administration compared to the model group, and as can be seen from B of fig. 5, the fecal water content of the model group (67.80 ± 0.90, # p <0.001) was significantly increased compared to the control group (53.65 ± 0.81) at the fourteenth day, and the fecal water content of the experimental group (58.76 ± 1.11, # p <0.001) was significantly decreased compared to the model group. The polysaccharide DOP0.2-S-3 is shown to reduce the water content in animal feces in an antibiotic-induced acute diarrhea model.

In summary, it can be seen from the test examples that the yam polysaccharide component DOP0.2-S-3 can reduce the symptoms of antibiotic-induced acute diarrhea, including maintaining the weight of the animal, reducing the number of times the animal defecates, and reducing the water content in the animal' S feces. The polysaccharide component DOP0.2-S-3 can be used as potential saccharide medicine for treating acute diarrhea and regulating intestinal flora disorder.